studies on protein kinase signalling pathway involved in
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STUDIES ON PROTEIN KINASESTUDIES ON PROTEIN KINASE
SIGNALLING PATHWAY INVOLVED INSIGNALLING PATHWAY INVOLVED IN
SCHIZOSACCHAROMYCES POMBESCHIZOSACCHAROMYCES POMBE
GENOME INSTITUTE OF SINGAPORE
M.SHANMUGA PRIYAA
M.Sc BIOTECHNOLOGY
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Comparative study, on the expression of proteinComparative study, on the expression of protein
kinase genes pertaining to the signaling pathway ofkinase genes pertaining to the signaling pathway of
the cell division cycle (cdc), ofthe cell division cycle (cdc), ofS.pombeS.pombe in both thein both the
wild and mutant strains is being investigated usingwild and mutant strains is being investigated using
micro array technology.micro array technology.
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Schizosaccharomyces pombe-fission yeast.
Pombe is derived from theSwahiliSwahiliword for beer.
Isolated in 1893 by Lindner frommilletbeerbeer..
The sixth model eukaryoticorganism whose genomegenome has beencompletely sequencedsequenced..
S.pombe is,
an unicellulareukaryoteeukaryote,,rod-shaped
2 - 3 m in diameter
7-14 m in length
WILD MUTANTWILD MUTANT
http://en.wikipedia.org/wiki/Swahilihttp://en.wikipedia.org/wiki/Swahilihttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Sequencedhttp://en.wikipedia.org/wiki/Genomehttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Swahili -
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Key players in the progression of the cell cycle are a group ofkinases.
CELL CYCLE :
INTERPHASE
G1-S-G2
M PHASE
PROPHASE,
METAPHASE,
ANAPHASE,
TELOPHASE.
Researches in cell cycle,helps to understand how cells grow and differentiate
as well as how the non-controlled growth results in cancerous cells.
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KINASES
Binds to cyclinscyclins-regulate kinase activity.
Inactive kinases-cdkscdks
Add phosphate group
Phosphorylation of proteins at different stages of the cell cycle is
necessary for the cycle to complete.
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GENE EXPRESSION:
ComplexComplex pattern of expression
Determines the characteristicscharacteristics of a cell and its rolerole in the organism.
Abnormal pattern- cancercancer..
MICROARRAY:
To assay gene expression within a single cell.
To compare gene expression in two different cell types or tissue samples.
To examine the expression of hundreds or thousands of genes at once.
STEPS:
1.Preparation ofDNA chipsDNA chips using known DNAs.2.Generation of a hybridization solution containing a mixture of
fluorescently labeled cDNAslabeled cDNAs (mutant type).
3.IncubationIncubation of the hybridization mixture containing fluorescently labeled cDNAs with
DNA chip.
4.Detection of bound cDNAs using llaserasertechnologytechnology and storing data in a computer.
5.Analyzing and interpreting the data using computationalcomputational methods.methods.
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Cell cultureCell culture
Total RNA extractionTotal RNA extraction
Reverse Transcriptional reactionReverse Transcriptional reaction
CDNA probe preparationCDNA probe preparation
Coupling CYS dyesCoupling CYS dyes
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Preparation of Micro
array slides.
Spotting of arrays
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Hybridization
Scanning
Yellow =equal expression
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The image analysis - 2 final intensity measurements for the red and greenThe image analysis - 2 final intensity measurements for the red and green
are obtained for every single spot.are obtained for every single spot.
GreenGreen represents - the expression of the control DNA (wildwild),
RedRed represents - the expression of the Sample DNA (referencereference),
yellowyellow represents - the expression of both the controlcontrol and samplesample
DNA.
BlackBlack represents - the expression of the neither the control norsample.
SCANNED SLIDESCANNED SLIDE
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CLUSTERED IMAGE PRODUCED USING GENE PIX PRO4.1CLUSTERED IMAGE PRODUCED USING GENE PIX PRO4.1
KO 42-pck1; KO 49-cdc2; KO 51-chk1; KO 55-fin1; KO 58-cmk1; KO 59-orb6;KO 42-pck1; KO 49-cdc2; KO 51-chk1; KO 55-fin1; KO 58-cmk1; KO 59-orb6;
KO 60-plo 1; KO 61-wis1; KO 62-spk 1; KO 65-cds1; KO 66-ssp1;KO 60-plo 1; KO 61-wis1; KO 62-spk 1; KO 65-cds1; KO 66-ssp1;KO 67-mek1; KO 75-kin1; KO 76-cdc7; KO 77-wee 1; KO 81-win 1;KO 67-mek1; KO 75-kin1; KO 76-cdc7; KO 77-wee 1; KO 81-win 1;
Each rowrow -- a gene Each columncolumn - a sample.
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The pattern of expression of Protein kinase mutant
strain
Orb6 (KO-59 A &B)
Each rowrow -- a gene
GreenGreen color - a particular
gene in the mutant is suppressedsuppressed
(down regulated).(down regulated).
RedRed color- a particular gene
in the mutant is induced (upinduced (up
regulated).regulated).
GREYGREY color - the missing data.
BLACKBLACK color - no expression
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The pattern of expression of Protein kinase mutant strain
Plo1 (KO-60 A &B)
GreenGreen color - a particular gene in
the mutant is suppressed.suppressed.
RedRed color - a particular gene
in the mutant is induced.induced.
GREYGREY color - the missing data.
BLACKBLACK color - no expression
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The pattern of expression of Protein kinase mutant strain
Wis1 (KO-61 A &B)
GreenGreen color - a particular gene in
the mutant is suppressed.suppressed.
RedRed color - a particular gene
in the mutant is induced.induced.
GREYGREY color - the missing data.
BLACKBLACK color - no expression
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The pattern of expression of Protein kinase mutant strain
Wee1 (KO-77 A &B)
GreenGreen color - a particular gene in
the mutant is suppressed.suppressed.
RedRed color - a particular gene
in the mutant is induced.induced.
GREYGREY color - the missing data.
BLACKBLACK color - no expression
T i f P t i ki t t
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Tree view of Protein kinase mutants
A short horizontal distanceA short horizontal distance - small intensity
differences between the two replicates.
A short vertical distance - the both replicates aresimilar.
The length of the branches indicates the distanceThe length of the branches indicates the distance
..
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Protein kinase mutants of
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Protein kinase mutants of
KO-58 (cmk1) KO-61(wis1) KO-67(mek1)
have correlated expression profiles with samebiological process and /orco-regulated.
Up-regulation (Induction) and Down regulation(Suppression) of different PK genes involved in thecell division cycle signaling pathway of PK mutants
strains helps us to understand the regulation of thecdc genes in Wild type S. pombe.
Comparison of Genomic expression programs within
and across different yeast can illuminate and explainthe physiological process within each organisms andcan reveal evolutionary differences in the regulationof genomic expression programs.
Thus micro array helps
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APPLICATIONAPPLICATION
- tissue/cell phenotype analysis by multiple gene expression assay,
- identification ofpathogens,
- genome-wide mutation survey
- diagnosis of genotypic polymorphisms and mutations.
- drug discovery,
- developmental biology
- molecular analysis of various diseases.
Thus micro array helps
- to assay gene expression within a sample orto compare
gene expression in two different cell types.
- to detect mutation orpolymorphism in a gene sequence
- to understand the growth and development
- to explore the underlying genetic causes ofmany humandiseases, especially cancer.
[cancer results from defects in fundamental cell regulatory
mechanisms, it is a disease that ultimately has to be understood at
the molecular and cellular levels.]
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